In order to optimize drug and oxygen delivery to solid tumors, we plan to investigate the mechanisms, which regulate tumor angiogenesis and microcirculation. This project will focus on the role of nitric oxide (NO) in tumor angiogenesis and vessel maturation. Our goal is to improve tumor blood flow and radiation response by judicious modulation of NO levels in tumors. Control of tumor blood flow has not been achieved due to the fact that tumor vessels are functionally impaired and heterogeneous with respect to diameter, length, tortuosity, and inter-capillary distance. However, transformation to a more "normal" functional phenotype in tumor blood vessels by suppressing NO may realize this goal. Effects of NO on cell proliferation and migration are different in vascular endothelial cells (induction) and smooth muscle cells (inhibition). We hypothesize that NO induces angiogenesis in tumors (Aim 1), but inhibits vessel maturation (Aim 2), and thus, chronic NO inhibition "normalizes" tumor vessels, making them sensitive to vasodilators. NO production will be manipulated by both pharmacological and genetic approaches. Isoform-selective NO synthase (NOS) inhibitors will clarify the relative contribution of each NOS isoform. With the use of NOS deficient mice, we will examine the contribution of host stromal cells. The tumors will include glioblastoma, melanoma, and breast carcinoma grown in orthotopic sites for primary and metastatic tumors. Intravital microscopy will allow us to monitor vessel density, diameter, tortuosity, blood flow rate, vascular permeability, and response to vasoactive agents. To study endothelial-mural cell interaction in vivo, mice expressing fluorescent protein in mural cells (alphaSMA-RFP to be developed in this project) and endothelial cells (TIE2-GFP) will be visualized by multiphoton laser-scanning microscopy. Finally, improvement of tumor oxygenation and response to radiation by a vasodilator and carbogen (O2 95 percent, CO2 5 percent) breathing after chronic NO suppression will be tested (Aim 3). The insight gained in this project will have significant implications for improving tumor treatment protocols. It will suggest a novel strategy (i.e. normalization of tumor vessels) to overcome some of the physiological barriers to the delivery of therapeutic agents to solid tumors and introduce a new paradigm to study cell-cell interaction in vivo.